Expression Of SF3B1 In Breast Cancer Tissues And The Effect Of Its Knockdown On Human ER Positive Breast Cancer Cell Function

Objective:To study the expression of splicing factor 3B subunit 1(SF3B1)in breast cancer tissues and the association with the clinicopathological parameters and investigate the effect of SF3B1 knockdown on proliferation,apoptosis,migration and invasion of estrogen receptor(ER)-positive breast cancer cells in vivo.Methods:Immunohistochemistry was used to assess the expression of SF3B1 in 110 breast cancer samples.ROC curve was used to determine the optimal pointcut value,which was divided into high expression group and low expression group and correlated with clinicopathological factors.SF3B1 knockdown in ZR-75-30 cells was performed by short hairpin RNA(shRNA)transfection,it was divided into knockdown group(SF3B1-sh1,SF3B1-sh2 group)and negative control group(ZR-75-30-NC group).The expression of SF3B1 in cells was detected by RT-qPCR and Western blot.Cell proliferation ability was determined by MTT and colony formation assay.Migration and invasion were determined by transwell assay.Flow cytometry was performed to investigate cell cycle and apoptosis.Statistical analysis was performed using SPSS 22.0.The measurement data were expressed as mean±standard deviation(Mean±SD).The mean comparison between the two groups was performed by t test.The comparison between groups was analyzed by one-way ANOVA.P < 0.05 was statistically significant.Results:A total of 110 cases were collected,including 38 cases of Luminal A type(34.5%),38 cases of Luminal B type(34.5%),14 cases of her2-enriched type(12.7%)and 20 cases of triple-negative breast cancer(18.2%).The tumor size was mostly concentrated in T1-T2,in 101 cases(91%),and the TNM stage was concentrated in I-II,in 68 cases(61.8%).Immunohistochemical results showed that SF3B1 is over expressed in breast cancer samples compared with matched normal tissue(P<0.01),this is particularly true in ER positive breast cancer.Through statistical analysis,thehigh expression of SF3B1 was associated with lymph node metastasis(P<0.05),no clear correlation was found with other clinicopathological factors,and no significant statistical difference was found between the subtypes.The endogenous high expres sion of SF3B1 in zr-75-30 cell lines was detected,and the knockdown sequences were imported into ZR-75-30 cell lines.RT-qPCR and western blot tests showed that the mRNA and protein levels of SF3B1 in the knockdown group were significantly lower than those in the negative control group.It suggested that we obtained cell lines with stable knockdown of SF3B1 gene.Colony formation experiments showed that the colony formation efficiency of the SF3B1 knockdown group was significantly reduced in the ZR-75-30 cell line.MTT showed that the absorbance of the SF3B1 knockdown group was significantly lower than that of the corresponding nega tive control group at 72 h,96h and 120h(P <0.05).Transwell invasion experiments showed that the invasion ability of the knockdown group cells was significantly reduced.The relative invasion rate was [(48.00% ± 4.91% or 40.1% ± 3.92%)vs.(155.10% ±2.31%),P <0.01].Transwell migration experiments showed that the numb er of migrated cells in the SF3B1 knockdown group was significantly lower than that in the negative control group [(107.00 ± 0.85 or 99.10 ± 2.88)vs.(153.00 ± 6.37),P <0.01].There was no significant difference in the cycle distribution between the SF3B1 knockdown group and the negative control group detected by flow cytometry,and the apoptosis rate in the knockdown group increased significantly [(9.75% ± 0.27% or 9.95%±0.47%)vs.(4.30% ± 0.22%),P <0.01].Conclusions:The expression of SF3B1 in breast cancer was significantly higher than that in normal tissue cells.The expression of SF3B1 was correlated with lymph node metastasis,but not with other clinicopathological factors.In ER positive breast cancer,SF3B1 gene knockdown can induce apoptosis;SF3B1 gene knockdown can reduce cell proliferation,colony formation,invasion and migration.SF3B1 may be an indicator of ER positive breast cancer clinical treatment and prognosis.However,the specific mechanism of action and the downstream shear events should be further studied to provide a new theoretical basis for the selection of molecular therapeutic targets of breast cancer.